Project Summary: Intracellular pathogens are a significant cause of morbidity and mortality worldwide. Some of these pathogens, such as Listeria monocytogenes, Francisella tularensis, Burkholderia psuedomallei, Rickettsia spp. and even Mycobacterium tuberculosis, spend at least a portion of their infectious cycle in the cytosol of their host cell. For many of these organisms, access to, and survival in the cytosol, is essential for virulence. Despite the ability of these diverse pathogens to survive and thrive in this niche, it has become clear that the cytosol is an inhospitable environment for non-cytosol adapted pathogens. However, little is known about the cell autonomous defenses that bacteria encounter in the cytosol and in turn the adaptations that cytosolic pathogens have evolved to overcome these stresses. L. monocytogenes is an important facultative intracellular pathogen that causes the disease Listeriosis. L. monocytogenes is also a powerful tool to understand complex host-pathogen interactions. In this proposal we will utilize a novel genetic screen to identify L. monocytogenes genes required for survival in the host cell cytosol. Mutants identified in this screen will be used to understand bacterial strategies for evasion of host defense, to determine the importance of cytosolic survival in virulence, and to define antimicrobial defense mechanisms in the cytosol. In addition to the genetic screen, we will take a targeted approach by characterizing a subset of genes already identified as essential for cytosolic survival and virulence. Specifically we will identify the unknown function of the bacterial small molecule metabolite 1,4-Dihydroxy-2-naphthoate (DHNA) in cytosolic survival and virulence. In addition, we will determine the mechanisms by which PrkA, a highly conserved PASTA kinase, functions as a master regulator of cell wall stress responses in the host cytosol. As one example, we will characterize how PrkA regulates the newly discovered uridyl transferase activity of the highly conserved protein YvcK and whether this regulation is conserved across species. Upon completion of these aims, we will have identified a collection of genes required for L. monocytogenes cytosolic survival and will have characterized two specific pathways (DHNA and PrkA) essential for L. monocytogenes virulence. These pathways may represent novel targets for therapeutic intervention. Indeed, the PASTA kinases are already the focus of novel antibiotic development by our labs and others, and as such, understanding their role in bacterial stress response and virulence is critical.